Protein tyrosine phosphate-1B (PTP-1B) deficient mice and uses thereof

ABSTRACT

The present invention provides mice that have had their PTP-1B genes disrupted by targeted homologous recombination. The mice have no detectable PTP-1B protein, yet appear to be physiologically normal. However, in the fed state on a normal diet, the mice have half the level of circulating insulin as their wild-type littermates. In glucose and insulin tolerance tests, the mice show an increased insulin sensitivity. When fed a high fat, high carbohydrate diet, the mice show a resistance to weight gain as compared to their wild-type littermates. Methods making the mice and cell lines derived from the mice are also provided. The present invention also provides methods of identifying inhibitors of the enzymatic activity of PTP-1B as well as inhibitors identified by such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a U.S. national filing under 35 U.S.C. §371 of PCT/CA99/00675,filed Jul. 23, 1999 and which published as WO 00/06712 on Feb. 10, 2000,and which claims the benefit of U.S. Provisional Patent Application No.60/093,975, filed on Jul. 24, 1998.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not applicable

REFERENCE TO MICROFICHE APPENDIX

Not applicable

FIELD OF THE INVENTION

The invention is directed to the field of transgenic mice containing adisrupted PTP-1B gene. The mice may contain a disruption in either oneor both copies of the PTP-1B gene. In the case of mice containing adisruption in both copies of the PTP-1B gene, such mice lack detectableexpression of PTP-1B protein.

BACKGROUND OF THE INVENTION

Protein tyrosine phosphatases (PTPases) are a large family oftransmembrane or intracellular enzymes that dephosphorylate substratesinvolved in a variety of regulatory processes (Fischer et al., 1991,Science 253:401-406). Protein tyrosine phosphatase-1B (PTP-1B) is a ˜50kd intracellular protein present in abundant amounts in various humantissues (Charbonneau et al., 1989, Proc. Natl. Acad. Sci. USA86:5252-5256; Goldstein, 1993, Receptor 3:1-15). Like other PTPases,PTP-1B has a catalytic domain characterized by the sequence motif(I/V)HCXAGXXR(S/T)G (SEQ.ID.NO.:1), containing arginine and cysteineresidues that are critical to the enzyme's activity (Streuli et al.,1990, EMBO J. 9:2399-2407; Guan et al., 1990, Proc. Natl. Acad. Sci. USA87:1501-1505; Guan & Dixon, 1991, J. Biol. Chem. 266:17026-17030). Theamino terminal 35 amino acid residues of PTP-1B localize the protein tothe endoplasmic reticulum (Frangioni et al., 1992, Cell 68:545-560).

Determining which proteins are substrates of PTP-1B has been ofconsiderable interest. One substrate which has aroused special interestis the insulin receptor. The binding of insulin to the insulin receptorresults in autophosphorylation of the receptor, most notably ontyrosines 1146, 1150, and 1151 in the kinase catalytic domain (White &Kahn, 1994, J. Biol. Chem. 269:1-4). This causes activation of theinsulin receptor tyrosine kinase, which phosphorylates the variousinsulin receptor substrate (IRS) proteins that propagate the insulinsignaling event further downstream to mediate insulin's variousbiological effects.

Seely et al., 1996, Diabetes 45:1379-1385 (Seely) studied therelationship of PTP-1B and the insulin receptor in vitro. Seelyconstructed a glutathione S-transferase (GST) fusion protein of PTP-1Bthat had a point mutation in the PTP-1B catalytic domain. Althoughcatalytically inactive, this fusion protein was able to bind to theinsulin receptor, as demonstrated by its ability to precipitate theinsulin receptor from purified receptor preparations and from whole celllysates derived from cells expressing the insulin receptor.

Ahmad et al., 1995, J. Biol. Chem. 270:20503-20508 used osmotic loadingto introduce PTP-1B neutralizing antibodies into rat KRC-7 hepatomacells. The presence of the antibody in the cells resulted in an increaseof 42% and 38%, respectively, in insulin stimulated DNA synthesis andphosphatidyinositol 3′ kinase activity. Insulin receptorautophosphorylation and insulin receptor substrate-1 tyrosinephosphorylation were increased 2.2 and 2.0-fold, respectively, in theantibody-loaded cells. The antibody-loaded cells also showed a 57%increase in insulin stimulated insulin receptor kinase activity towardexogenous peptide substrates.

Until the present invention, studies of the interaction of PTP-1B andthe insulin receptor were limited to studies conducted on cell-freepreparations of PTP-1B or in cultured cell lines. Therefore, suchstudies did not address the issue of whether PTP-1B activity affects theregulation of the insulin receptor in a way that results inphysiological effects on glucose metabolism, triglyceride metabolism, orweight gain in living mammals. Because of the complexity of theregulation of the insulin receptor and its interactions with proteinssuch as PTP-1B, there is a need to study this regulation in anenvironment that is as close as possible to that of a living mammal. Theknockout mice of the present invention are useful in helping to meetthis need. The knockout mice of the present invention also are useful inthat they provide an animal model that can be used in the design andassessment, in a living mammal, of compounds that modulate insulinreceptor activity.

SUMMARY OF THE INVENTION

The present invention provides mice that have had their PTP-1B genesdisrupted by targeted homologous recombination. When both copies oftheir PTP-1B genes are disrupted, the mice have no detectable PTP-1Bprotein, yet appear to be physiologically normal. However, in the fedstate, the mice have slightly lower glucose levels and half the level ofcirculating insulin as their wild-type littermates. In glucose andinsulin tolerance tests, the mice show increased insulin sensitivity.When fed a high fat, high carbohydrate diet, the mice, although muchmore insulin-sensitive than wild-type controls, are obesity-resistant.

Methods of making the mice and cell lines derived from the mice are alsoprovided.

The present invention also provides methods of identifying inhibitors ofthe enzymatic activity of PTP-1B as well as inhibitors identified bysuch methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Gene targeting of the PTP-1B locus. A) Genomic organization ofthe mouse PTP-1B gene and design of targeting vector. Exons areindicated by boxes and exon 6, which contains the active site cysteine,is unfilled. Restriction endonuclease recognition sites are abbreviatedas follows: B=BamHI; E=EcoRI; H=HindIII; Xb=XbaI; Xh=Xhol. Bottom is thegenomic structure of a homologous recombination event. B) Representativegenomic Southern blot using the PTP-1B 3′ probe on tail DNA digestedwith BamHI from a heterozygous cross resulting in wild type (+/+),heterozygous (+/−), and homozygous (“null”) (−/−) PTP-1B offspring. C)PTP-1B immunoblot analysis of liver membrane samples from PTP-1B (+/+),PTP-1B (+/−), and PTP-1B (−/−) mice.

FIG. 2. Glucose and insulin levels in ad libitum-fed and overnightfasted PTP-1B (+/+), PTP-1B (+/−), and PTP-1B (−/−) mice fed on a normal(i.e., non-high fat, non-high carbohydrate) diet. A) Glucose and B)Insulin levels were determined as described in the Examples herein. Thenumber of mice in the fed groups A and B were (n=19-21) and in thefasted group A (n=8-10) and in B (n=6). The values are given as themeans ± s.e.m. Statistical analysis was done with a two-tailed unpairedStudent's t-test, *, (P=0.06), ** (P≦0.01).

FIG. 3. Glucose and insulin tolerance tests in PTP(+/+) (wild-type) andPTP(−/−) (null) mice fed on a normal (i.e., non-high fat, non-highcarbohydrate) diet. A) Glucose tolerance was performed on male mice10-14-weeks-old (n=11−12); ▪=wild-type; =null. B) Insulin tolerance onmale mice 10-14-weeks-old (n=5−6); ▪=wild-type; =null. The data arepresented as the means ± s.e.m. Statistical analysis was done with atwo-tailed unpaired Student's t-test and compared to wild-type, *(P≦0.05), ** (P≦0.02).

FIG. 4. Disruption of the murine PTP-1B gene results in hyper andprolonged tyrosine phosphorylation of the insulin receptor (IR) in micefed on a normal (i.e., non-high fat, non-high carbohydrate) diet. A)Representative immunoblot showing the time course of tyrosinephosphorylation on the IR β-subunit after insulin challenge in liver forthe times indicated for PTP-1B (+/+) and PTP-1B (−/−) mice.Quantification of the insulin receptor β-subunit phosphotyrosine levelsfrom immunoblots was performed by densitometry. Data is presented bysetting the 1 min phosphotyrosine insulin receptor β-subunit level foreach animal to 100% and the subsequent 5 min level for the same animalrelative to this value. The results are from five PTP-1B (−/−) andPTP-1B (+/+) mice each, from three separate experiments. B) Immunoblotshowing the phosphorylation level of the IR β-subunit in muscle frominsulin-treated PTP-1B (+/+) and PTP-1B (−/−) mice. The quantified datafrom the immunoblot (n=3) is given in arbitrary desitometer units withthe 2 min time point from the wild type mice set at 100. C) IRS-1immunoblot from muscle of insulin treated PTP-1B (+/+) and PTP-1B (−/−)mice, 2 minutes post-injection (n=3). In the SDS polyacrylamide (7.5%)gels (15 cm×15 cm) used for the immunoblots, IRS-1 ran as a diffuse 185kD band. Data are presented as the means ± s.e.m. Statistical analysiswas done with a two-tailed unpaired Student's t-test comparing in A) the5 minute to the 1 mute time point values and in B) the PTP-1B (−/−) mice2 minute and 6 minute time point values to the respective values of thePTP-1B (+/+) mice, * (P≦0.05).

FIG. 5. Weight gain in A) male and B) female null knockout, heterozygousknockout, and wild-type mice fed a high fat, high carbohydrate diet. A)male mice; ♦=wild-type, n=7; ▪=heterozygotes, n=9; ▴=nulls, n=8. B)female mice; ♦=wild-type, n=9; ▪=heterozygotes, n=9; ▴=nulls, n=8.

FIG. 6. Glucose and insulin challenge in mice fed a high fat, highcarbohydrate diet demonstrating the development of insulin resistance inPTP-1B wild type mice and the relative lack of insulin resistance inPTP-1B knockout mice. A) Glucose and B) insulin tolerance tests of malemice on a high fat, high carbohydrate diet. ♦=wild-type, n=7;▪=heterozygotes, n=7 (A) or 8 (B); ▴=nulls, n=7. C) Insulin-stimulatedinsulin receptor tyrosine phosphorylation level in muscle in mice fed ahigh fat, high carbohydrate diet (PTP-1B (−/−) and PTP-1B (+/+), n=2;PTP-1B (+/−), n=3). Quantitation of immunoblots were performed asdescribed in FIG. 4. * (P≦0.05)

FIG. 7. Tyrosine phosphorylation level of the insulin receptor in fatafter insulin challenge in PTP-1B (+/+) and PTP-1B (−/−) mice. Levels ofinsulin receptor phosphotyrosine in fat from mice on normal diet A) andhigh fat diet B). Quantitation of immunoblots were performed asdescribed in FIG. 4 setting the 3 min time point of the wild type miceto 100. The data represent the average of two mice from each group ±s.e.m.

FIG. 8. Induction of uncoupling protein-1 and brown adipocytes in whiteadipose tissue of PTP-1B (−/−) mice. A) Northern blot analysis of UCP-1and UCP-2 mRNA expression in abdominal fat of wild type (n=2) and PTP-1B(−/−) mice (n=2). B) Histology of inguinal white adipose tissue (IWAT)from wild type and PTP-1B (−/−) mice showing induction of multilocularadipocytes in PTP-1B (−/−) IWAT. C) Histology of interscapular brownadipose tissue (IBAT) from wild type and PTP-1B (−/−) mice. Note thelarger fat droplets in wild type IBAT adipocytes compared to IBAT ofPTP-1B (−/−) mice.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of this invention:

A “transgenic mouse” is any mouse containing one or more cells bearinggenetic information altered or received, directly or indirectly, bydeliberate genetic manipulation at a subcellular level, such as bytargeted homologous recombination, microinjection, or infection withrecombinant virus. The term “transgenic mouse” is not intended toencompass classical cross-breeding or in vitro fertilization, but ratheris meant to encompass mice in which one or more cells are altered by orreceive a recombinant DNA molecule. A transgenic mouse can have thegenetic alteration or genetic information introduced into a germ linecell, thereby conferring the ability to transfer the genetic informationto offspring. If such offspring possess some or all of that alterationor genetic information, then they, too, are transgenic mice.

A “knockout mouse” is a mouse in which the expression of a preselectedgene has been suppressed or eliminated by introducing into the genomicDNA of the mouse a new DNA sequence that serves to disrupt some portionof the DNA sequence of the preselected gene. A knockout mouse may haveboth copies of the preselected gene disrupted, in which case it is ahomozygous or “null” knockout mouse. A knockout mouse may have only asingle copy of the preselected gene disrupted, in which case it is a“heterozygous” knockout mouse.

One approach to the problem of determining the role of a particular genein a biochemical pathway or a disease state is to selectively inactivatethe native wild-type gene in totipotent ES cells and then generatetransgenic mice using those ES cells. Such transgenic mice, having thatparticular gene inactivated, are known as “knockout mice.” The use ofgene-targeted ES cells in the generation of gene-targeted transgenicknockout mice is described in, e.g., Thomas et al., 1987, Cell51:503-512, and is reviewed elsewhere (Frohman et al., 1989, Cell56:145-147; Capecchi, 1989, Trends in Genet. 5:70-76; Baribault et al.,1989, Mol. Biol. Med. 6:481-492).

Techniques are available to inactivate or alter any genetic region tovirtually any mutation desired by using targeted homologousrecombination to insert specific changes into chromosomal genes.Generally, use is made of a “targeting vector,” i.e., a plasmidcontaining part of the genetic region it is desired to mutate. By virtueof the homology between this part of the genetic region on the plasmidand the corresponding genetic region on the chromosome, homologousrecombination can be used to insert the plasmid into the genetic region,thus disrupting the genetic region. Usually, the targeting vectorcontains a selectable marker gene as well.

In comparison with homologous extrachromosomal recombination, whichoccurs at frequencies approaching 100%, homologous plasmid-chromosomerecombination was originally reported to only be detected at frequenciesbetween 10⁻⁶ and 10⁻³ (Lin et al., 1985, Proc. Natl. Acad. Sci. USA82:1391-1395; Smithies et al., 1985, Nature 317:230-234; Thomas et al.,1986, Cell 44:419-428). Nonhomologous plasmid-chromosome interactionsare more frequent, occurring at levels 10⁵-fold (Lin et al., 1985, Proc.Natl. Acad. Sci. USA 82:1391-1395) to 10²-fold (Thomas et al., 1986,Cell 44:419-428) greater than comparable homologous insertion.

To overcome this low proportion of targeted recombination in murine EScells, various strategies have been developed to detect or select rarehomologous recombinants. One approach for detecting homologousalteration events uses the polymerase chain reaction (PCR) to screenpools of transformant cells for homologous insertion, followed byscreening individual clones (Kim et al., 1988, Nucleic Acids Res.16:8887-8903; Kim et al., 1991, Gene 103:227-233). Alternatively, apositive genetic selection approach has been developed in which a markergene is constructed which will only be active if homologous insertionoccurs, allowing these recombinants to be selected directly (Sedivy etal., 1989, Proc. Natl. Acad. Sci. USA 86:227-231). One of the mostpowerful approaches developed for selecting homologous recombinants isthe positive-negative selection (PNS) method developed for genes forwhich no direct selection of the alteration exists (Mansour et al.,1988. Nature 336:348-352; Capecchi, 1989, Science 244:1288-1292;Capecchi, 1989, Trends in Genet. 5:70-76). The PNS method is moreefficient for targeting genes which are not expressed at high levelsbecause the marker gene has its own promoter. Nonhomologous recombinantsare selected against by using the Herpes Simplex virus thymidine kinase(HSV-TK) gene and selecting against its nonhomologous insertion withherpes drugs such as gancyclovir (GANC) or FIAU (1-(2-deoxy2-fluoro-B-D-arabinofluranosyl)-5-iodouracil). By thiscounter-selection, the percentage of homologous recombinants in thesurviving transformants can be increased.

The present invention provides mice that have had their PTP-1B genesdisrupted by targeted homologous recombination. In the case of mice thathave had both copies of their PTP-1B genes disrupted (“null” mice), themice have no detectable PTP-1B protein, yet appear to be physiologicallynormal. However, in the fed state, the null mice have slightly lowerglucose levels and half the level of circulating insulin as theirwild-type littermates. In glucose and insulin tolerance tests, the nullmice show an increased insulin sensitivity. Furthermore,hyperphosphorylation of the insulin receptor is evident in the liver andmuscle of the null mice injected with insulin when compared to wild-typemice treated similarly. These results indicate that PTP-1B is involvedin the insulin signaling pathway in living mammals and has a role in thedephosphorylation and hence inactivation of the insulin receptor.

The PTP-1B knockout mice of the present invention are useful as sourcesof cell lines that do not contain functional PTP-1B protein. Such celllines can be transfected with the human PTP-1B gene to produce celllines expressing human PTP-1B free from interference caused by theexpression of endogenous mouse PTP-1B. Such cell lines can be used inassays to identify activators or inhibitors of human PTP-1B.Accordingly, in addition to PTP-1B knockout mice, the present inventionprovides cell lines derived from the PTP-1B knockout mice of the presentinvention. Such cell lines can be produced by methods well known in theart (Williams et al., 1988, Mol. Cell. Biol. 8:3864-3871; Aaronson &Todaro, 1968, J. Cell. Physiol. 72:141-148: Jenkins et al., 1984, Nature312:651-654).

The PTP-1B knockout mice of the present invention are useful as negativecontrols in assays that monitor the effects of pharmaceuticals thatmodulate glucose metabolism, triglyceride metabolism, or weight gainthrough an effect of the pharmaceuticals on PTP-1B in wild-type mice.The use of PTP-1B knockout mice as negative controls in such assaysallows one to determine that an effect on glucose metabolism,triglyceride metabolism, or weight gain caused by a pharmaceutical inwild-type mice that is suspected of being caused by the action of thepharmaceutical on PTP-1B activity is actually so caused.

The PTP-1B knockout mice of the present invention are useful in assaysto identify weak agonists of the insulin receptor. Since these knockoutmice lack PTP-1B, they lack an important element involved in dampeningthe signal of the insulin receptor. Thus, in the absence of PTP-1B inthe knockout mice, weak agonists of the insulin receptor can beidentified where the effects of those weak agonists would have beenmissed in the presence of PTP-1B. Such weak agonists can be used as leadcompounds which can be modified by medicinal chemistry to developstronger, pharmacologically useful, agonists of the insulin receptor.

The PTP-1B knockout mice of the present invention are useful forstudying the role of the insulin receptor in various aspects ofmetabolism or physiology. For example, the mice can be monitored todetermine if the loss of PTP-1B has any effect on their longevity. In C.elegans, mutations in the gene daf-2 affect longevity. Daf-2 is ahomolog of the mammalian insulin receptor.

Db/db mice develop many complications, such as peripheral neuropathiesand myocardial disease, similar to those of humans with diabetes. ThePTP-1B knockout mice of the present invention can be crossed with db/dbmice in order to better study the relationship between PTP-1B activityand diabetes. The PTP-1B knockout mice of the present invention can beused in a similar manner with other chemically or genetically induceddiabetic mouse models.

It will be of interest to investigate the effects of glucose levellingdrugs such as thiazolidinediones or sulfonylureas in the PTP-1B knockoutmice of the resent invention.

In view of the demonstration herein that PTP-1B regulation of theinsulin receptor may have a role in obesity, it will be of interest toinvestigate the effects of leptin, the melanocortin-4 receptor, theneuropcptide Y₅ receptor, and other molecular species that have beenimplicated in weight control in PTP-1B knockout mice.

A variety of methods can be used to produce the knockout mice of thepresent invention. One method involves introducing a transgene intotarget cells that are then incorporated into blastocyts that areimplanted into pseudopregnant female mice. A type of target cell fortransgene introduction is the embryonal stem cell (ES). ES cells may beobtained from pre-implantation embryos cultured in vitro (Evans et al.,1981, Nature 292: 154-156; Bradley et al., 1984, Nature 309: 255-258;Gossler et al., 1986, Proc. Natl. Acad. Sci. USA 83: 9065-9069;Robertson et al., 1986, Nature 322, 445-448; Wood et al., 1993, Proc.Natl. Acad. Sci. USA 90: 4582-4584). Transgenes can be efficientlyintroduced into ES cells by standard techniques such as DNA transfectionor by retrovirus-mediated transduction. The resultant transformed EScells can thereafter be combined with blastocysts from a mouse.Following implantation of the blastocyts into pseudopregnant fostermothers, the introduced ES cells can thereafter colonize the embryosthat develop from the blastocyts and contribute to the germ line of theresulting chimeric mice (Jaenisch, 1988, Science 240: 1468-1474).

Another method that can be used to produce the knockout mice of thepresent invention involves microinjecting the transgene into the maleproinucleus of a ferilized egg (Brinster et al., 1981, Cell 27:223;Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:5016; Sterwart etal., 1982, Science 217:1046-1048; Townes et al., 1985, EMBO J. 4:1715).The microinjected transgene integrates into the DNA of the malepronucleus of the fertilized egg. The ferilized egg is then implantedinto a recipient female mouse and allowed to develop. If this procedureis successful, the resulting embryo will contain the transgene in allits cells. Occasionally, the fertilized egg will divide before thetransgene integrates into the genome. In such cases, chimeric embryoswill be produced. Such chimeric embryos will contain the transgene insome, but not all, of their cells.

The present invention provides a method of producing a mouse, at leastsome of whose cells contain an altered gene encoding an altered form ofprotein tyrosine phosphatase-1B (PTP-1B), the altered gene having beentargeted to replace the wild-type PTP-1B gene in the mouse, the methodcomprising:

(a) providing an altered gene encoding an altered form of PTP-1Bdesigned to target the PTP-1B gene of mouse embryonic stem (ES) cells;

(b) introducing the altered gene encoding an altered form of PTP-1B intomouse ES cells;

(c) selecting ES cells in which the altered gene encoding an alteredform of PTP-1B has disrupted the wild-type PTP-1B gene;

(d) injecting the ES cells from step (c) into mouse blastocyts;

(e) implanting the blastocysts from step (d) into a pseudopregnantmouse;

(f) allowing the blastocytsts to develop into embryos and allowing theembryos to develop to term in order to produce a mouse at least some ofwhose cells contain an altered gene encoding an altered form of PTP-1B.

In the case where the mice produced by the above-described methodcontain germ cells with the altered gene encoding an altered form ofPTP-1B, the mice may be mated to produce mice all of whose somatic cellsas well as germ cells contain the altered gene encoding an altered formof PTP-1B. Mice having germ cells containing the altered gene encodingan altered form of PTP-1B can be mated to produce homozygous, or “null,”mice that contain disruptions in both copies of their PTP-1B genes andthus have no detectable PTP-1B activity.

The knockout mice of the present invention have altered glucose and fatmetabolism compared to wild-type mice. The effect of the disruption ofthe PTP-1B gene in the knockout mice of the present inventiondemonstrates that altering the activity of PTP-1B can modulate insulinsignaling in vivo, i.e., in a living mammal. The knockout mice of thepresent invention also demonstrate that altering the activity of PTP-1Bcan have an effect on weight gain. These results suggest that inhibitionof PTP-1B may be beneficial in the treatment of Type II diabetes(non-insulin dependent diabetes, NIDDM) and obesity. The presentinventors are the first to demonstrate an effect of PTP-1B on suchaspects of fuel metabolism in a living mammal as levels of blood glucoseand triglycerides, or weight gain. Prior to the present invention, itwas not predictable that the interaction of PTP-1B and the insulinreceptor that was seen in purified enzyme preparations or in tissueculture cells could be extrapolated to effects such as these, which canonly be studied in living mammals. Accordingly, before the presentinvention, it was not reasonably predictable that one could useinhibitors of the interaction betweeen PTP-1B and the insulin receptorto modulate levels of blood glucose or triglycerides in a living mammal,or control obesity, since the relevance of experiments done in tissueculture to the regulation of fuel metabolism in living mammals was notclear.

Prior to the work presented herein, it was not thought that such asimple change as knocking out PTP-1B alone would have such dramaticeffects as those observed by the present inventors. This is because itwas known that insulin receptor action is regulated in a complex manner.Among the various classes of proteins involved in this regulation,several protein tyrosine phosphatases (PTPases) alone were known to beinvolved. For example, Kulas et al., 1995, J. Biol. Chem. 270:2435-2438demonstrated that insulin receptor activity is negatively regulated bythe PTPase LAR. Others had shown that the PTPase SH2-PTP (aka Syp)positively regulates insulin activity (Xiao et al., 1994, J. Biol. Chem.269:21244-21248; Milarski et al., 1994, J. Biol. Chem. 269:21239-21243;Yamauchi et al., 1995, Proc. Natl. Acad. Sci. USA 92:664-668; Noguchi etal., 1994, Mol. Cell. Biol. 14:6674-6682). Hashimoto et al., 1992. J.Biol. Chem. 267:13811-123814 demonstrated that a number of proteintyrosine phosphatases can dephosphorylate the insulin receptor in vitroas efficiently as PTP-1B. Numerous other reports demonstrate that PTPsother than PTP-1B can also dephosphorylate the activated insulinreceptor (Jacob et al., 1998, J. Biol. Chem. 273:4800-4809: Chiarugi etal., 1997, Biochem. Biophys. Res. Commun. 238:676-682; Moller et al.,1995, J. Biol. Chem. 270:23126-23131).

Given such a complex regulatory mechanism as that which governs theactivity of the insulin receptor, one would have expected that knockingout a single component of that mechanism in a living mammal would haveproduced little effect, since that single component either would havebeen quantitatively insignificant in itself, or since it would have beenexpected that other components of the regulatory mechanism would havecompensated for the lack of the knocked-out component, restoring thebalance of the insulin receptor activity to its normal state. See, e.g.,Ahmad et al., 1995, J. Biol. Chem. 270:20503-20508, at page 20508, whosum up the results of their studies as follows: “[I]nsulin signalling isbalanced at multiple levels by a number of PTPases . . . ” Despite theseexpectations, the present invention demonstrates that it is possible tocontrol insulin receptor activity in a living mammal throughmodification of the activity of PTP-1B. Accordingly, based upon theresults demonstrated by the knockout mice of the present invention, itis now feasible to identify inhibitors of the enzymatic activity ofPTP-1B that will be useful in modulating the activity of the insulinreceptor in living mammals. Such inhibitors should have utility in thetreatment of Type II diabetes and obesity.

Prior to the present invention, it was believed that while suchinhibitors of PTP-1B might possibly have desirable effects on theinsulin receptor, they would not be pharmacologically useful since itwas believed that PTP-1B had many essential roles in addition to itsrole in modulating insulin receptor activity. Therefore, it was believedthat in addition to possible desirable effects on the insulin receptor,inhibitors of the enzymatic activity of PTP-1B would have too manydeleterious side effects to be pharmacologically useful. The prior artthus taught that inhibitors of the binding of PTP-1B to the insulinreceptor might be useful (since such inhibitors would have effectsspecific to the insulin receptor) but inhibitors of the enzymaticactivity of PTP-1B would not be useful (since such inhibitors would havemore general effects). See, e.g., International Patent Publication WO97/32595, at page 11, line 36, to page 12, line 7: “It is preferable toaffect binding [of PTP-1B to the insulin receptor] rather thanphosphatase activity since phosphatase activity in general is essentialto the cell.” Another example of how the prior art taught away from theuse of inhibitors of the enzymatic activity of PTP-1B can be seen inclaim 1 of U.S. Pat. No. 5,726,027, which reads: “A method fordetermining whether a composition inhibits protein tyrosine phosphatase1B (PTP1B) binding to phosphorylated insulin receptor rather thanphosphatase activity, said method comprising . . . ”

In view of the demonstration by the present invention that PTP-1Bknockout mice are physiologically normal, with the exception of alteredglucose and triglyceride metabolism, as well as altered weight gainpatterns when fed a high fat, high carbohydrate diet, it is clear thatthe prior art was mistaken when it assumed that inhibitors of PTP-1Benzymatic activity would lack utility. The present invention makes itclear that inhibitors of PTP-1B enzymatic activity are likely to haveutility for the treatment of Type II diabetes and in the control ofobesity. Accordingly, the present invention provides methods ofidentifying inhibitors of the enzymatic activity of PTP-1B as well asinhibitors so indentified by such methods.

The present invention provides a method of identifying inhibitors of theenzymatic activity of PTP-1B comprising:

(a) providing an enzymatically active preparation of PTP-1B;

(b) measuring the enzymatic activity of PTP-1B in the enzymaticallyactive preparation of PTP-1B in the presence and in the absence of asubstance suspected of being an inhibitor of the enzymatic activity ofPTP-1B;

where a decrease in the enzymatic activity of PTP-1B in the presence ascompared to the absence of the substance indicates that the substance isan inhibitor of the enzymatic activity of PTP-1B.

Of course, the above-described method may be practiced in such a mannerthat step (b) is carried out with not just a single substance suspectedof being an inhibitor of the enzymatic activity of PTP-1B, but ratherwith a plurality of substances suspected of being inhibitors of theenzymatic activity of PTP-1B. An example of the practice of the methodin this manner would be the screening of a library of compounds, e.g., acombinatorial library, against the enzymatically active preparation ofPTP-1B. In such cases, typically only a small fraction of the substancesin the library will be found to be inhibitors of the enzymatic activityof PTP-1B. If the library is large, it may be divided into convenientlysmall portions of substances for use in step (b).

The present invention provides a method of identifying inhibitors of theenzymatic activity of the PTP-1B protein comprising:

(a) transfecting a cell with DNA encoding the human PTP-1B protein;

(b) culturing the cells of step (a) under conditions such that PTP-1Bprotein is produced;

(c) measuring the enzymatic activity of the PTP-1B protein in thetransfected cells in the presence and in the absence of a substancesuspected of being an inhibitor of the enzymatic activity of the PTP-1Bprotein;

where a decrease in the enzymatic activity of the PTP-1B protein in thepresence as compared to the absence of the substance indicates that thesubstance is an inhibitor of the enzymatic activity of the PTP-1Bprotein.

The above-described method may be practiced in such a manner that step(c) is carried out with not just a single substance suspected of beingan inhibitor of the enzymatic activity of the PTP-1B protein, but ratherwith a plurality of substances suspected of being inhibitors of theenzymatic activity of the PTP-1B protein. An example of the practice ofthe method in this manner would be the screening of a library ofcompounds, e.g., a combinatorial library. In such cases, typically onlya small fraction of the substances in the library will be found to beinhibitors of the enzymatic activity of the PTP-1B protein. If thelibrary is large, it may be divided into conveniently small portions ofsubstances for use in step (c).

The cells of step (a) may be prokaryotic or eukaryotic, mammalian oramphibian, bacterial, or yeast. Cell lines derived from mammalianspecies which are suitable and which are commercially available, includebut are not limited to, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M(ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL70), COS-1 (ATCC CRL 1650). COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL61),3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I(ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171). In apreferred embodiment, the cells are cells that have been derived from aPTP-1B null knockout mouse of the present invention.

Transfection is meant to include any method known in the art forintroducing DNA sequences encoding the PTP-1B protein into the cells.For example, transfection includes calcium phosphate or calcium chloridemediated transfection, lipofection, infection with a retroviralconstruct containing DNA sequences encoding the PTP-1B protein, andelectroporation.

Methods of measuring the enzymatic activity of the PTP-1B protein may becarried out by any methods known in the art. For example, thedephosphorylation of the insulin receptor by PTP-1B may be measured, asin Maegawa et al., 1995, J. Biol. Chem. 270:7724-7730. See also Huyer etal., 1997, J. Biol. Chem. 272:843-851. Other methods include: measuringthe activity of PTP-1B in intact cells by, e.g., immunoprecipitating theenzyme and measuring its activity with a variety of artificialsubstrates such as FDP, MUP, p-nitrophenylphosphate, phosphotyrosylpeptides, or ³²P/³³P-labeled phosphopeptides or phosphoproteins. Inaddition, the activity of PTP-1B can be measured by following thephosphotyrosyl status not only of the insulin receptor but also of anyother substrates of PTP-1B. Also, where the insulin receptor remainsphosphorylated for long times, as in the liver, or where the insulinreceptor is hyperphosphorylated, as in muscle, the phosphorylation stateof proteins involved in the insulin cascade and the biochemical effectsof that cascade will be increased. Thus, one can indirectly follow theactivity of PTP-1B in intact cells by measuring such factors as: glucosetransport, glycogen synthesis, amino acid transport, protein synthesis,phosphorylation status of insulin receptor substrates, P13 kinaseactivity, Akt kinase activity, etc.

In step (c) of the above-described method, the transfected cells may beused intact or they may be first lysed and certain fractions of thecells, or partially or wholly purified preparations of the PTP-1Bprotein from the cells, may be used.

The substances identified by the above-described methods are especiallyuseful when those substances are specific inhibitors of the enzymaticactivity of PTP-1B, i.e., when those substances do not also inhibit theactivity of another protein tyrosine phosphatase. Accordingly, it willgenerally be worthwhile to take the inhibitors identified by theabove-described methods and further screen them against such otherprotein tyrosine phosphatases as, e.g., LAR, syp, CD45, etc.

The above-described methods of identifying inhibitors of the enzymaticactivity of PTP-1B can be modified so as to become methods fordetermining whether the substances identified by the above-describedmethods can be used to treat Type II diabetes and associatedcomplications or to control obesity. This would entail taking thesubstances identified as inhibitors of the enzymatic activity of PTP-1Band determining whether those substances modulate glucose level ortriglyceride levels in a mammal, such as a mouse, rat, or human, orwhether those substances prevent or diminish obesity in a mammal, suchas a mouse, rat, or human, that is fed a high fat, high carbohydratediet.

Accordingly, the present invention provides a method of determiningwhether a substance modulates glucose or triglyceride levels in a mammalthat comprises:

(a) providing an enzymatically active preparation of PTP-1B;

(b) measuring the enzymatic activity of PTP-1B in the enzymaticallyactive preparation of PTP-1B in the presence and in the absence of asubstance suspected of being an inhibitor of the enzymatic activity ofPTP-1B, thus identifying a substance that is an inhibitor of theenzymatic activity of PTP-1B;

where a decrease in the enzymatic activity of PTP-1B in the presence ascompared to the absence of the substance indicates that the substance isan inhibitor of the enzymatic activity of PTP-1B;

(c) administering the substance that is an inhibitor of the enzymaticactivity of PTP-1B to a mammal;

(d) measuring the blood glucose level or triglyceride levels of themammal in step (c) and comparing the blood glucose level or triglyceridelevels of the mammal in step (c) with the blood glucose level ortriglyceride levels of a mammal that has not been administered thesubstance that is an inhibitor of the enzymatic activity of PTP-1B;

where a difference in the blood glucose level or triglyceride levels ofthe mammal in step (c) as compared with the blood glucose level ortriglyceride levels of the mammal that has not been administered thesubstance that is an inhibitor of the enzymatic activity of PTP-1Bindicates that the substance modulates glucose or triglyceride levels ina mammal.

In a particular embodiment, the mammal is a mouse or rat. In anotherembodiment, the mammal is a human.

The present invention provides substances identified by theabove-described method. Such substances are expected to have utility inthe treatment of Type II diabetes and associated complications inhumans.

The present invention provides a method of determining whether asubstance regulates obesity in a mammal that comprises:

(a) providing an enzymatically active preparation of PTP-1B;

(b) measuring the enzymatic activity of PTP-1B in the enzymaticallyactive preparation of PTP-1B in the presence and in the absence of asubstance suspected of being an inhibitor of the enzymatic activity ofPTP-1B, thus identifying a substance that is an inhibitor of theenzymatic activity of PTP-1B;

where a decrease in the enzymatic activity of PTP-1B in the presence ascompared to the absence of the substance indicates that the substance isan inhibitor of the enzymatic activity of PTP-1B;

(c) administering the substance that is an inhibitor of the enzymaticactivity of PTP-1B to a mammal;

(d) measuring the weight gain of the mammal in step (c) when the mammalof step (c) is fed a high fat, high carbohydrate diet and comparing theweight gain of the mammal in step (c) with the weight gain of a mammalfed a high fat, high carbohydrate diet that has not been administeredthe substance that is an inhibitor of the enzymatic activity of PTP-1B;

where a difference in the weight gain of the mammal in step (c) ascompared with the weight gain of the mammal that has not beenadministered the substance that is an inhibitor of the enzymaticactivity of PTP-1B indicates that the substance regulates obesity in amammal.

In a particular embodiment, the mammal is a mouse or rat. In anotherembodiment, the mammal is a human.

The present invention provides substances identified by theabove-described method. Such substances are expected to have utility incontrolling obesity in humans.

The present invention provides a method of determining whether asubstance modulates glucose or triglyceride levels in a mammal thatcomprises:

(a) transfecting a cell with DNA encoding the human PTP-1B protein;

(b) culturing the cells of step (a) under conditions such that PTP-1Bprotein is produced;

(c) measuring the enzymatic activity of the PTP-1B protein in thetransfected cells in the presence and in the absence of a substancesuspected of being an inhibitor of the enzymatic activity of the PTP-1Bprotein;

where a decrease in the enzymatic activity of the PTP-1B protein in thepresence as compared to the absence of the substance indicates that thesubstance is an inhibitor of the enzymatic activity of the PTP-1Bprotein;

(d) administering the substance that is an inhibitor of the enzymaticactivity of PTP-1B to a mammal;

(e) measuring the blood glucose level or triglyceride levels of themammal in step (d) and comparing the blood glucose level or triglyceridelevels of the mammal in step (d) with the blood glucose level ortriglyceride levels of a mammal that has not been administered thesubstance that is an inhibitor of the enzymatic activity of PTP-1B;

where a difference in the blood glucose level or triglyceride levels ofthe mammal in step (d) as compared with the blood glucose level ortriglyceride levels of the mammal that has not been administered thesubstance that is an inhibitor of the enzymatic activity of PTP-1Bindicates that the substance modulates glucose or triglyceride levels ina mammal.

In a particular embodiment, the mammal is a mouse or rat. In anotherembodiment, the mammal is a human.

The present invention provides a method of determining whether asubstance regulates obesity in a mammal that comprises:

(a) transfecting a cell with DNA encoding the human PTP-1B protein;

(b) culturing the cells of step (a) under conditions such that PTP-1Bprotein is produced;

(c) measuring the enzymatic activity of the PTP-1B protein in thetransfected cells in the presence and in the absence of a substancesuspected of being an inhibitor of the enzymatic activity of the PTP-1Bprotein;

where a decrease in the enzymatic activity of the PTP-1B protein in thepresence as compared to the absence of the substance indicates that thesubstance is an inhibitor of the enzymatic activity of the PTP-1Bprotein;

(d) administering the substance that is an inhibitor of the enzymaticactivity of PTP-1B to a mammal;

(e) measuring the weight gain of the mammal in step (d) when the mammalof step (d) is fed a high fat, high carbohydrate diet and comparing theweight gain of the mammal in step (d) with the weight gain of a mammalfed a high fat, high carbohydrate diet that has not been administeredthe substance that is an inhibitor of the enzymatic activity of PTP-1B;

where a difference in the weight gain of the mammal in step (d) ascompared with the weight gain of the mammal that has not beenadministered the substance that is an inhibitor of the enzymaticactivity of PTP-1B indicates that the substance regulates obesity in amammal.

In a particular embodiment, the mammal is a mouse or rat. In anotherembodiment, the mammal is a human.

The present invention includes inhibitors of the enzymatic activity ofPTP-1B that have been identified by the above-described methods. Suchinhibitors have many uses. For example, such inhibitors can be used in amethod of treating obesity comprising administering an inhibitor of theenzymatic activity of PTP-1B to an obese mammal. Such inhibitors canalso be used in a method of treating Type II diabetes and associatedcomplications comprising administering an inhibitor of the enzymaticactivity of PTP-1B to a person with Type II diabetes.

Such inhibitors are generally combined with pharmaceutically acceptablecarriers before use. Examples of such carriers and methods offormulation of pharmaceutically acceptable compositions containinginhibitors and carriers can be found in Remington's PharmaceuticalSciences. To form a pharmaceutically acceptable composition suitable foreffective administration, such compositions will contain an effectiveamount of the inhibitor.

Therapeutic or prophylactic compositions are administered to anindividual in amounts sufficient to treat or prevent obesity or Type IIdiabetes. The effective amount can vary according to a variety offactors such as the individual's condition, weight, sex and age. Otherfactors include the mode of administration. The appropriate amount canbe determined by a skilled physician.

Compositions can be used alone at appropriate dosages. Alternatively,co-administration or sequential administration of other agents can bedesirable.

The compositions can be administered in a wide variety of therapeuticdosage forms in conventional vehicles for administration. For example,the compositions can be administered in such oral dosage forms astablets, capsules (each including timed release and sustained releaseformulations), pills, powders, granules, elixirs, tinctures, solutions,suspensions, syrups and emulsions, or by injection. Likewise, they canalso be administered in intravenous (both bolus and infusion),intraperitoneal, subcutaneous, topical with or without occlusion, orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts.

Advantageously, compositions can be administered in a single daily dose,or the total daily dosage can be administered in divided doses of two,three or four times daily. Furthermore, compositions can be administeredin intranasal form via topical use of suitable intranasal vehicles, orvia transdermal routes, using those forms of transdermial skin patcheswell known to those of ordinary skill in that art. To be administered inthe form of a transdermal delivery system, the dosage administrationwill, of course, be continuous rather than intermittent throughout thedosage regimen.

The dosage regimen utilizing the compositions is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal, hepatic andcardiovascular function of the patient; and the particular compositionthereof employed. A physician or veterinarian of ordinary skill canreadily determine and prescribe the effective amount of the compositionrequired to prevent, counter or arrest the progress of the condition.Optimal precision in achieving concentrations of composition within therange that yields efficacy without toxicity requires a regimen based onthe kinetics of the composition's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a composition.

The following non-limiting examples are presented to better illustratethe invention.

EXAMPLE 1 Construction of the Targeting Vector

In order to construct the targeting vector, the mouse PTP-1B gene wascloned from a 129/Sv mouse genomic library and characterized. The mousePTP-1B gene was isolated by screening a Lambda FIX II 129/SvJ mousegenomic library (Stratagene, La Jolla, Calif.) using the human (GenBankaccession no. M317324; see also Chernoff et al., 1990, Proc. Natl. Acad.Sci. USA 87:2735-2739) and mouse (GeneBank accession no. M97590; seealso Miyasaka et al., 1990, Bioichem. Biophys. Res. Comm. 185:818-825)PTP-1B cDNAs as probes. Three overlapping λ clones were isolated and thegenomic organization and exon sequences of the mouse PTP-1B gene weredetermined. The three λ clones contained the majority of the PTP-1B geneexcept for the 5′ flanking region and the first 190 bp of the cDNA (FIG.1A). The gene is composed of at least 9 exons spanning greater than 20kb. A targeting vector (FIG. 1A) was made by deleting genomic sequencesthat included exon 5 and exon 6 (which contains the tyrosine phosphataseactive site cysteine 215) and replacing the deleted sequences with theneomycin-resistance gene. This was accomplished by the cloning of theneo marker gene driven by the PGK promoter (PGK-neo) into the Smal siteof pBluescript KS⁺ (Statagene, La Jolla, Calif.) (pneoKS). The 5.5 kbHind III/EcoRI mouse PTP-1B genomic fragment which is just 5′ to exon 5was then inserted into HindIII/EcoRI digested pneoKS. This vector wasthen digested with XbaI and NotI and ligated with the 1.6 kb XbaI/XhoImouse PTP-1B genomic fragment which is just 3′ of exon 6 and a XhoI/NotIHSV-tk gene driven by the PGK promoter fragment. The resulting targetingvector (pTARGET) was linearized by HindIII digestion.

EXAMPLE 2 Production of Knockout Mice

The targeting vector was electroporated into 129/Sv embryonic stem cells(J1) and G418 resistant colonies were selected as described previously(You-Ten et. al , 1997, J. Exp. Med. 186:683-693). J1 cells are meant tobe illustrative only. Other embryonic stem cell lines are suitable aswell, e.g., ES-D3 cells (ATCC catalogue no. CRL-1934). Coloniesresistant to G418 were analyzed for homologous recombination by BamHIdigestion of genomic DNA followed by Southern blotting and hybridizationwith probe A. Probe A is the 800 bp XhoI/BamHI fragment shown as “3′probe” in FIG. 1A. Approximately 2% of the resistant colonies underwenta homologous recombination event. Two of these G418 resistant ES cellclones were then used for microinjection into Balb/c blactocysts asdescribed previously (You-Ten et. al. , 1997, J. Exp. Med. 186:683-693).Germline transmission was obtained for each line and F1 heterozygoteswere mated to produce animals homozygous for the PTP-1B mutation, i.e.,null mice. Genotyping was performed by Southern blotting (FIG. 1B). Adouble band of 3.4 and 2.7 kb is seen in PTP-1B heterozygotic mice and asingle band of 2.7 kb is seen in PTP-1B null mice. Immunoblot analysisof liver microsomes revealed that PTP-1B protein was absent in PTP-1Bnull mice (FIG. 1C). These results demonstrate that the PTP null miceare lacking the PTP-1B enzyme.

EXAMPLE 3 Glucose and Insulin Levels in Knockout Mice Fed a Normal Diet

Glucose and insulin levels were measured in fasted and fed mice on anormal, i.e., non-high fat, non-high carbohydrate, diet (FIG. 2). In thefed state the null mice had a significant (P≦0.01) 13% reduction inblood glucose levels compared to wild-type mice, whereas theheterozygotes had an 8% reduction when compared to wild type (FIG. 2A).Surprisingly, the null mice also had circulating insulin levels thatwere about half that of wild-type fed animals (FIG. 2B). These resultssuggest that the fed PTP-1B-deficient mice are much more sensitive toinsulin, resulting in greater glucose lowering in response to much lessinsulin. In the fasted state, there were no significant differences inglucose or insulin levels among the wild-type, null, and heterozygotemice. However, there was a substantial reduction in triglyceride levelsin the fasted state in the PTP-1B null and heterozygote knockout mice ascompared to wild-type mice. The triglyceride levels in the fasted nullmice (0.86±0.18 mM/L) were about 50% lower than in the wild-type mice(1.84±0.76 mM/L) and were 20% lower in heterozygotes (1.43±0.44). SeeTable 1.

TABLE 1 PTP-1B^(+/+) PTP-1B^(+/−) PTP-1B^(−/−) Diet Normal High FatNormal High Fat Normal High Fat Glucose 6.1 ± 0.3 8.1 ± 0.6 6.2 ± 0.37.3 ± 0.6 6.3 ± 0.3  7.0 ± 0.4† (mM/L) Trigly- 1.84 ± 0.76 2.41 ± 0.191.43 ± 0.44 2.44 ± 0.32  0.86 ± 0.18* 1.46 ± 15*  cerides (mM/L) Insulin0.30 ± 0.02 0.98 ± 0.32 ND 0.97 ± 0.30 0.33 ± 0.08  0.45 ± 0.14* (ng/ml)Table 1. Fasting glucose, triglyceride and insulin levels of male PTP-1B(−/−), wild type, and heterozygote littermates fed a normal or a highfat, high carbohydrate diet. The values are given as the means ± s.e.m.Statistical analysis was done with a two-tailed unpaired Student'st-test and compared to wild type. ND, not determined. †(P = 0.1), *(P <0.05) (n = 6-10)

Triglyceride levels in the fed state were unaffected. These datademonstrate that loss of PTP-1B affects glucose and triglyceridehomeostasis in the knockout mice, for the first time stronglyimplicating PTP-1B in the insulin signaling pathway in an intact mammal.

Insulin sensitivity in PTP-1B null and wild-type mice fed a normal dietwas further examined by performing oral glucose and intraperitonealinsulin tolerance tests. Administration of a bolus of glucose to PTP-1Bnull mice resulted in a more rapid clearance of glucose than thatobserved for wild type mice (FIG. 3A). There was a more pronouncedhyperglycemia in the wild type animals at all time points post-gavagewhen compared to PTP-1B null mice. Increased insulin sensitivity wasalso observed upon injection of insulin (FIG. 3B). In both null andwild-type mice, hypoglycemia was evident at 30 and 60 minutes postinjection. However, whereas wild type glucose levels approached normallevels after 120 minutes, the PTP-1B null mice remained hypoglycemic(P<0.02).

EXAMPLE 4 Tyrosine Phosphorylation of the Insulin Receptor and InsulinReceptor Substrate-1 in Knockout Mice

To determine whether PTP-1B affects the phosphorylation of the insulinreceptor in vivo, i.e., in a living mammal, the phosphotyrosine level ofthe insulin receptor was measured in both muscle and liver of knockoutand wild-type mice after insulin challenge. Insulin was injected as abolus into the inferior vena cava and tissue samples were taken atvarious times post-injection in order to determine the time course ofinsulin receptor dephosphorylation. The insulin receptor β-subunit wasthen immunoprecipitated from the membrane fraction of tissue homogenatesand immunoblotted with an anti-phosphotyrosine antibody to determine thelevel of phosphorylation of the insulin receptor. The blot was thenstripped and reprobed with a C-terminal β-subunit antibody to determinethe amount of the β-subunit on the blot in order to normalize thephosphotyrosine signal to the amount of β-subunit. In both null andwild-type mice, in either liver or muscle, the level of insulin receptorphosphorylation in the absence of insulin was very low (FIG. 4A and B).Insulin treatment of wild-type mice resulted in a dramatic increase inthe level of insulin receptor tyrosine phosphorylation in the liver(FIG. 4A) which fell to about 50% of the 1 min level by 5 min (FIG. 4A)post-injection (P<0.05). This time course of insulin receptorphosphorylation in the liver has been previously observed in rats with at_(½) of 6 min (Rothenberg et al., 1991, J. Biol. Chem. 266:8302-8311).However, in null mice treated similarly, the kinetics of insulinreceptor phosphorylation in the liver were significantly different thanthose observed for the wild-type mice. The level of insulin receptorphosphorylation was the same for both null and wild-type mice after 1min post injection, but unlike the wild-type mice, the level of tyrosinephosphorylation in null mice after 5 min post-injection did not decreaseand was virtually identical to the 1 min level (P<0.05). The sustainedhyperphosphorylation of the insulin receptor in the null mice suggeststhat the insulin receptor would also remain activated for a much longerperiod in these mice. However, the most striking effect on insulinreceptor hyperphosphorylation was observed in the muscle of the nullmice. Analysis of the phosphotyrosine levels of the insulin receptor inmuscle samples from insulin treated null mice revealed that there wasabout a 40% increase in the absolute level of phosphorylation comparedto wild-type muscle levels (P<0.05) (FIG. 4B). Unlike the level inliver, the level of insulin receptor phosphorylation in muscle did notdecrease over the time course of the experiment in either null orwild-type mice. It is unlikely that the hyperphosphorylation of theinsulin receptor observed in the muscle is due to an overexpression ofthe insulin receptor in null mice, since no detectable difference in thelevel of insulin receptor expression was observed in either wild-type ornull mice as determined by immunoblotting of total tissue lysates. Thissubstantial increase in insulin receptor phosphorylation in the muscleand the sustained phosphorylation of the insulin receptor in the liverof null mice are most likely responsible for the increased insulinsensitivity in these mice. This would also tend to suggest that the illvivo substrate for PTP-1B, especially in muscle, is the activatedinsulin receptor.

In order to confirm that the hyperphosphorylation of the insulinreceptor in the muscle of insulin treated null mice also translates intoincreased kinase activity, the phosphotyrosine level of the insulinreceptor substrate-1 (IRS-1) was examined in the 2 minutespost-injection samples (FIG. 4C). IRS-1 was hyperphosphorylated inmuscle of insulin treated null mice compared to wild-type mice (P<0.05).Furthermore, the time course of IRS-1 dephosphorylation in liver hasbeen found to be even more rapid than the insulin receptor, returning tobaseline levels after only 2-3 minutes (Rothenberg et al., 1991, J.Biol. Chem. 266:8302-831 1). Nevertheless, hyperphosphorylation ofIRS-1, to the same level as the 2 minute time point, was also evident inthe 6 minute post-injection null muscle samples.

EXAMPLE 5 Obesity Resistance in PTP-1B Knockout Mice Fed a High Fat,High Carbohydrate Diet

Wild-type mice that are fed a high fat, high carbohydrate diet becomeobese and develop obesity-induced insulin resistance (Uysal et al.,1997, Nature, 389:610-614). Male and female PTP-1B knockout mice(heterozygotes as well as null), 7-8 weeks old, were fed a high fat;high carbohydrate diet (50% calories from fat, 5,286 kcal kg-1, Bio-ServF3282 mouse diet, Bioserv, Frenchtown, N.J.). As controls, wild-typemice were fed the same diet. After 10 weeks on this diet, both male andfemale wild type littermates became obese, whereas PTP-1B (−/−) andPTP-1B (+/−) mice were significantly protected from diet-induced obesity(FIG. 5). The starting weights of the animals put on the diet were notsignificantly different (male, +/+, 27.6±1.4; +/−, 28.5±1.2; −/−,26.3±1.2 g; and female, +/+, 22.1±0.8; +/−, 22.2±0.8; −/−21.5±0.8 g)while the final weight of the wild type mice when compared to bothPTP-1B heterozygotes and null animals (male, +/+, 41.4±1.3; +/−,37.2±2.0; −/−, 33.5±1.6 g and female, +/+, 33.3±1.7; +/−, 27.3±1.3;−/−27.2±1.4 g) showed a significant difference (P<0.05 wild type versusheterozygotes or null except for male wild type versus heterozygotcwhich was P=0.1). The amount of food consumed by all groups of animalswhile on the diet did not differ, suggesting that changes in theexpression levels of PTP-1B (heterozygotes have about half the level ofPTP-1B expression as wild type, FIG. 1C) can affect development ofdietary induced obesity.

In order to examine the effect the high fat, high carbohydrate diet hadon insulin sensitivity in PTP-1B (+/+),PTP-1B (+/−), and PTP-1B (−/−)mice, fasting glucose and insulin levels, as well as glucose and insulintolerance tests, were performed on all groups of animals (Table 1,above, and FIG. 6; only male values are presented, female values gaveessentially the same result). The high fat, high carbohydrate dietproduced a 30% increase in fasting glucose levels and a three foldincrease in fasting insulin levels in the PTP-1B (+/+) mice (Table 1).In contrast, the PTP-1B (−/−) animals maintained lower glucose andinsulin levels while on the high fat diet, levels that are notsignificantly different from the normal diet values (Table 1). Theseresults indicate that the high fat diet resulted in insulin resistancein the wild type littermates, but not in the PTP-1B (−/−) mice. The fatfed PTP-1B heterozygotes also showed elevated fasting insulin levels buthad fasting glucose levels that were lower than wild type (Table 1).Increased insulin sensitivity was also observed in the PTP-1B (−/−) micecompared to their wild type littermates in both glucose and insulintolerance tests (FIGS. 6A and B), with PTP-1B (+/−) mice appearing toshow intermediate sensitivity. The difference in insulin sensitivitybetween the PTP-1B (−/−) and PTP-1B (+/+) mice as measured by glucoseand insulin tolerance became even more evident on the high fat diet(compare FIGS. 3A and B to FIGS. 6A and B). This was also the case whenthe tyrosine phosphorylation level of the insulin receptor in muscle wasmeasured after insulin challenge in the mice on the high fat, highcarbohydrate diet (FIG. 6C). It has been shown that a high fat diet cancause an obesity-related reduction in insulin receptor signaling inmuscle and fat tissue (Uysal et al., 1997, Nature, 389:610-614). In FIG.4B, insulin stimulation of mice fed a normal diet caused about a 40%increase in the phosphotyrosine level of the insulin receptor in muscleof PTP-1B (−/−) compared to PTP-1B (+/+) mice. The high fat dietincreased this difference in insulin sensitivity between the wild typeand PTP-1B (−/−) mice to the extent that the PTP-1B (−/−) mice haveabout a 4 fold higher insulin receptor phosphorylation level than wildtype, whereas the PTP-1B (+/−) mice show an intermediate level (about 2fold higher than wild type) (FIG. 6C). The expected phenotype, namelyobesity and insulin resistance, was observed for the PTP-1B (+/+) micefed a high fat diet. In contrast, both PTP-1B (+/−) and PTP-1B (−/−)mice presented an unexpected phenotype in that they were resistant tothe development of obesity. Insulin sensitivity was maintained in thePTP-1B (−/−) mice, while the PTP-1B (+/−) mice showed an intermediatesensitivity compared to the PTP-1B wild type and null mice.

The above-described results in mice fed a high fat, high carbohydratediet demonstrate that PTP-1B knockout mice remain insulin-sensitive onthis diet. In fact, they are much more insulin-sensitive than theirwild-type littermates. Thus, it would have been expected that PTP-1Bknockout mice, when fed on a high fat, high carbohydrate diet, would beat least as susceptible to obesity as wild-type mice, if not more so,since their increased insulin sensitity would have been expected toinduce increased lipogenesis in the knockout mice. However, theexperiments described below show that just the opposite occurs.

The weight of the PTP-1B knockout and wild-type mice fed a high fat,high carbohydrate diet, as well as the amount of food the mice consumed,was measured each week. There was essentially no difference in foodconsumption between wild-type, heterozygote, and null mice. After tenweeks of being on the high fat, high carbohydrate diet, wild type micehad about a 50% weight gain; heterozygote and null mice both had onlyabout a 25-30% weight gain. Thus, the knockout mice have about half theweight gain of wild-type mice when fed a high fat, high carbohydratediet. See FIG. 5. These results indicate that PTP-1B plays a role inobesity and that inhibitors of the enzymatic activity of PTP-1B will beharmacologically useful in the control of obesity.

EXAMPLE 6 Induction of Uncoupling Protein in White Adipose Tissue ofPTP-1B^(−/−) Mice

To investigate the reason for the obesity resistance in the PTP-1B (−/−)mice, we measured fasting triglyceride levels in mice on either the highfat or normal diet. The PTP-1B (−/−) mice on either diet hadsignificantly lower triglyceride levels than wild type and heterozygousmice. The PTP-1B (+/−) mice had slightly lower triglyceride levels onthe normal diet compared to wild type mice but showed no differencecompared to wild type when on the high fat, high carbohydrate diet. Thisresult indicates that the loss of PTP-1B had an effect on fatmetabolism. Accordingly, the phosphotyrosine level of the insulinreceptor in adipose tissue was examined after insulin challenge inanimals fed a normal or high fat diet. Contrary to liver and muscle,which showed hyperphosphorylation of the insulin receptor, thereappeared to be a hypophosphorylation of the insulin receptor in fat fromPTP-1B (−/−) mice compared to wild type, suggesting that adipose tissueof PTP-1B (−/−) mice may to some extent be insulin resistant (FIG. 7A).Support for this comes from the fact that the high fat fed wild typemice which are insulin resistant now have insulin receptorphosphorylation levels in adipose tissue basically equivalent to that ofthe PTP-1B (−/−) mice (FIG. 7B). Thus PTP-1B-deficient mice appear toshow tissue specific insulin sensitivity. Liver and muscle are moresensitive, whereas fat tissue appears to be resistant, compared to wildtype mice.

Altered insulin signaling in the fat tissue of the PTP-1B (−/−) mice islikely one of the factors that contributes to the obesity resistanceobserved in these animals. Insulin action on adipocytes results indecreases in cAMP levels and stimulation of lipogenesis (Manganiello etal., 1996, Curr. Top. Cell. Regul. 34:63-100). In the PTP-1B deficientmice, adipocytes have a reduced insulin response and consequently may beresistant to fat formation. It has been well documented that increasingthe activity of protein kinase A (PKA) in adipocytes either byincreasing cAMP levels through the action of β adrenergic receptoractivity or by altering the PKA regulatory subunits through geneknockout can result in obesity resistance due to the induction ofuncoupling protein-1 (UCP-1). UCP-1 is a mitochondrial protontranslocator that uncouples the oxidation of fatty acids in BAT. Thisresults in the energy derived from the breakdown of fatty acids beingdissipated as heat instead of ATP formation, thus raising the body'sresting metabolic rate (Himms-Hagen & Ricquier, 1998, “Brown AdiposeTissue” in Handbook of Obesity (eds Bray, G. A, Bouchard, C. & James, W.P. T.) pages 415-441 (Marcel Dekker Inc., New York; Cummings et al.,1996, Nature 382:622-626). If the loss of PTP-1B activity has affectedcAMP levels in the adipose tissue of PTP-1B (−/−) mice due to alteredinsulin receptor activity in adipose tissue, then this may account forthe obesity resistance phenotype observed in the PTP-1B (−/−) mice.Northern blot analysis was performed to investigate whether UCP wasinduced in the white adipose tissue (WAT) of PTP-1B wild type and nullmice (normal diet). Induction of UCP-1 mRNA in abdominal WAT of twoseparate PTP-1B (−/−) mice was apparent, whereas it was undetectable inPTP-1B (+/+) WAT (FIG. 8A). UCP-2 mRNA levels were unchanged betweenwild type and PTP-1B (−/−) mice (FIG. 8A). UCP-1 mRNA is only expressedin brown adipocytes and its expression in a white adipose depotindicates the induction of brown adipose in this fat depot (Ghorbani etal., 1997, Biochem. Pharmacol. 54:121-131). Histological analysis ofinguinal WAT from wild type mice showed the expected typical largeunilocular adipocyte (FIG. 8B). In contrast, the inguinal WAT fromPTP-1B (−/−) mice contained much smaller unilocular adipocytes and, moreimportantly, revealed the presence of many multilocular adipocytes notnormally found in this fat depot (FIG. 8B). The multilocular adipocyteis characteristic of brown adipose, consistent with the UCP-1 mRNAexpression observed in the PTP-1B (−/−) WAT. Immunological stainingshowed that the multilocular cells in the inguinal WAT from PTP-1B (−/−)mice stained positive for UCP protein. Examination of interscapular BAT(IBAT) revealed that the PTP-1B (+/+) mice contained adipocytes withlarger lipid droplets than found in PTP-1B (−/−) mice (FIG. 8C).

EXAMPLE 7 Glucose and Insulin Measurements

Blood was collected from the orbital sinus of anesthetized mice andserum was prepared. Serum glucose levels were determined using a Vitros250 analyzer and radioimmunoassay (Linco. St.Charles, Mo.) was used tomeasure insulin levels.

Glucose tolerance was performed after an overnight fast byadministration of 1 g/kg of glucose by gavage and blood collected att=0, 30, 60 and 120 min in anesthetized male mice (10-14-weeks-old).Plasma was prepared and frozen until use and serum glucose levelsdetermined. Insulin tolerance tests were performed after an overnightfast by intraperitoneal injection of 0.75 U/kg of regular human insulin(Eli Lilly, Indianapolis. Ind.). For both glucose tolerance and insulintolerance tests, blood was collected from the tail of mice and one dropof blood was placed on the One Touch Strip glucose assay system andglucose levels were monitored to the corresponding One Touch Basic(Lifescan Canada Ltd., Burnaby, British Columbia, Canada).

EXAMPLE 8 In vivo Analysis of Insulin Receptor Phosphorylation

After an overnight fast, mice were anesthetized, the abdominal cavityexposed, and 5 units of regular human insulin (Eli Lilly, Indianapolis,Ind.) or saline was injected as a bolus into the inferior vena cava(Araki et al., 1994, Nature 372:186-190). One minute after injection asmall piece of liver was excised and immediately frozen in liquidnitrogen. Approximately 2 min after the injection a piece of quadricepsmuscle and abdominal fat was removed and quick frozen. This was againrepeated at 5, 6, and 7 min post-injection for liver, muscle, and fat,respectively and the mice were then sacrificed before recovery.

EXAMPLE 9 Immunoprecipitation and Immunoblot Analysis

Immunoblot analysis of PTP-1B expression in liver membrane fractions (25μg/lane) of wild-type, heterozygotic, and null mice was perfomed usingan N-terminal specific (amino acids 43-56) PTP-1B rabbit polyclonalantibody (UBI). The blot was developed using enhanced chemiluminescence(NEN). Immunoprecipitation of the insulin receptor β-subunit wasperformed as follows. The tissue, either liver, fat, or muscle, washomogenized on ice in 50 mM Tris pH 7.5, 150 mM NaCl, 1 mMpyrophosphatc, 100 uM pervanadate (a potent PTPase inhibitor; Huyer et.al., 1997, J. Biol. Chem. 272:843-851) and a protease inhibitor cocktail(Boehringher Mannheim). A membrane fraction was prepared bycentrifugation at 100,000×g for 1 h and protein concentrationdetermined. Two hundred μg of liver or muscle membrane protein, or 100μg of fat membrane protein, was solubilized in immunoprecipitationbuffer (RIPA) (150 mM NaCl, 10 mM phosphate buffer pH 7.5 1% NP-40, 1%Na deoxycholate, 0.1% SDS) and immunoprecipitation of the insulinreceptor β-subunit was carried out overnight at 4° C. using 1 μg of theanti insulin receptor antibody (C-19) (Santa Cruz Biotechnology, SantaCruz, Calif.) followed by a 60 min incubation of 50 μl of a 50% slurryof protein G sepharose (Pharmacia Biotech). The sample was washed 3times in 1 ml of RIPA buffer and samples were loaded on an 8% SDS PAGE.The samples were transferred onto PVDF membrane and immunodetection ofphosphotyrosine was performed using the anti-phosphotyrosine 4G10 horseradish peroxidase coupled antibody (Upstate Biotech) according to themanufacturer's protocol. The same blot was stripped in 62.5 mM Tris pH6.7, 2% w/v SDS, 100 mM β-mercaptoethanol for 30 min at 55° C., washed,and reprobed with the anti insulin β-subunit Rb (C-19, Santa CruzBiotechnology, Santa Cruz, Calif.). The phosphotyrosine signal andβ-subunit levels were then quantiated by densitometry (MolecularDynamics) and phosphotyrosine levels normalized to the amount ofβ-subunit present in each sample. Immunoprecipitation of IRS-1 wasperformed with two IRS-1 rabbit polyclonal antibodies (C-20, C-terminusspecific and A-19, N-terminus specific, Santa Cruz Biotechnology, SantaCruz, Calif.) using the cytosolic fraction from muscle of insulintreated mice, as described above.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1 1 11 PRT Artificial Sequence SITE 1 Xaa = Ile or Val 1 Xaa His Cys XaaAla Gly Xaa Xaa Arg Xaa Gly 1 5 10

What is claimed:
 1. A transgenic mouse whose genome is homozygous for adisrupted PTP-1B gene, such that said mouse has no detectable PTP-1B,and wherein said mouse exhibits increased insulin sensitivity ascompared to wild-type mice.
 2. The mouse of claim 1 wherein the PTP-1Bgene is disrupted by the insertion of a plasmid comprising a selectablemarker gene.
 3. The mouse of claim 2 wherein the PTP-1B gene isdisrupted by the insertion of pTARGET.
 4. The mouse of claim 1 whereinthe mouse has about half the level of circulating insulin in the fedstate as compared to wild-tape mice.
 5. The mouse of claim 1 wherein themouse has about 13% of the level of blood glucose in the fed state ascompared to wild-type mice.
 6. A cell line established from thetransgenic mouse of claim 1, wherein the cells have no detectablePTP-1B.
 7. A transgenic mouse whose genome is homozygous for a disruptedPTP-1B gene, such that said mouse has no detectable PTP-1B, and whereinsaid mouse exhibits resistance to diet induced obesity as compared towild-type mice.
 8. The mouse of claim 7 wherein the PTP-1B gene isdisrupted by the insertion of a plasmid comprising a selectable markergene.
 9. The mouse of claim 7 wherein the PTP-1B gene is disrupted bythe insertion of pTARGET.
 10. The mouse of claim 7 wherein the mouse hasabout half the weight gain of wild-type mice when fed a high fat, highcarbohydrate diet.
 11. A transgenic mouse whose genome is homozygous fora disrupted PTP-1B gene, such that said mouse has no detectable PTP-1B,and wherein said mouse exhibits a phenotype selected from the groupconsisting of mice having about half the level of circulating insulin inthe fed state as compared to wild-type mice, having about 13% of thelevel of blood glucose in the fed state as compared to wild-type miceand having about half the weight gain when fed a high fat, highcarbohydrate diet as compared to wild-type mice.
 12. A method ofproducing a mouse whose genome is homozygous for a disrupted PTP-1Bgene, such that said mouse has no detectable PTP-1B, the methodcomprising: (a) providing a gene encoding an altered form of PTP-1Bdesigned to target the PTP-1B gene of mouse embryonic stem (ES) cells,wherein the form comprises a disruption such that no detectable PTP-1Bis produced; (b) introducing the gene encoding an altered form of PTP-1Binto mouse ES cells; (c) selecting ES cells in which the altered geneencoding an altered form of PTP-1B has disrupted the wild-type PTP-1Bgene; (d) injecting the ES cells from step (c) into mouse blastocysts;(e) implanting the blastocysts from step (d) into a pseudopregnantmouse; (f) allowing the blastocysts to develop into embryos and allowingthe embryos to develop to term in order to produce a mouse homozygousfor a disrupted PTP-1B gene.
 13. A transgenic mouse whose genome isheterozygous for a disrupted PTP-1B gene, wherein said disrupted gene ina homozygous state produces a mouse that has no detectable PTP-1B, andwherein said mouse exhibits increased insulin sensitivity as compared towild-type mice.